DNA helicases are multi-subunit, macromolecular machines that use energy derived from ATP hydrolysis to unwind duplex segments to drive essential cellular processes such as DNA replication, recombination, and repair. Like its eukaryotic and archaeal counterparts, the bacterial replicative helicase, DnaB, plays many important roles in the highly-regulated process of DNA replication, including formation of a bidirectional replication fork. To assist in fork formation, the hexameric ring of DnaB must first be opened and deposited onto the single-stranded DNA regions of a melted replication origin, a process that depends on the initiator protein, DnaA, and the DnaB loading partner, DnaC. How specific protein-protein and protein-nucleotide interactions facilitate the loading of two DnaB hexamers in an orientation- and strand-specific manner remains an outstanding and important question. Using biochemical and structural methods, the mechanism of helicase loading will be elucidated by determining the precise roles of replication initiator and helicase loading proteins, and their use of ATP, in this process. Specifically, the role of DnaA- and DnaC-dependent interactions with DnaB in promoting the orientation-specific helicase loading onto each DNA strand of a melted replication origin will be investigated (Aim 1). Recent data suggest that DnaA and DnaC have distinct, but complementary, functions in loading two DnaB hexamers onto DNA. To test this idea, a plasmid-based, biochemical DNA footprinting assay will be used, together with site-directed mutagenesis, to probe how specific interactions between DnaA, DnaB, and DnaC differentially contribute to the loading of two helicase hexamers in opposing directions on the two complementary strands of a melted origin. This approach will allow for detection of DNA strand-specific effects on helicase loading. Additionally, the molecular basis for the interaction of DnaB helicase with DnaA and DnaC will be defined using a structural approach (Aim 2). DnaA and DnaC are both multi-domain proteins in which critical functional modules are connected by flexible tethers. As this type of molecular configuration can interfere with structural investigations, the minimal regions of both DnaA and DnaC sufficient to interact with DnaB will be established, and these fragments will be used as entryways to determine co-crystal structures of each with DnaB. High-resolution structures of these co-complexes will provide a molecular understanding of the interactions necessary to support helicase loading, and whether DnaA or DnaC binding alters the conformation of DnaB. PUBLIC HEALTH RELEVANCE: Among all domains of life, genetic stability is dependent upon the regulated process of DNA replication initiation, an event that requires proper loading of two copies of helicase for formation of a bidirectional replication fork. Accumulating data suggests that the general process of replication initiation is conserved among all organisms, including the need for replication initiator and helicase loader functionalities, however, the similarities and differences between these systems have not been established. This proposal uses bacterial replication as a model system to elucidate the complimentary yet distinct roles of initiator and helicase loader proteins in helicase loading, and will provide the molecular details necessary for developing replication initiation as a target for new antibacterial and chemotherapeutic agents.